The present invention refers to a new process for the synthesis of polypeptide 1, comprising the following amino acid units in the structure, namely: L-alanine, L-glutamic acid, L-lysine and L-tyrosine randomly arranged in the polypeptide 1; of which Glatiramer Acetate is a representative example.
Glatiramer Acetate is a synthetic polypeptide analog of myelin basic protein (MBP), which is a natural component of the myelin sheath. It is also defined in the Physicians' Desk Reference, 56th Edition 2002 as consisting of acetate salts of synthetic polypeptides, containing four naturally occurring amino acids: namely, L-glutamic acid, L-alanine, L-tyrosine and L-lysine with an average molar fraction of 0.141, 0.427, 0.095 and 0.338 respectively. The average molecular weight is 4,700–11,000 daltons.
Interest in Glatiramer Acetate as an immunotherapy agent for multiple sclerosis stems from the 1950's observations that myelin components such as MBP prevent or arrest experimental allergic encephalomyelitis, a disease resembling multiple sclerosis (U.S. Pat. No. 3,849,550). Recently, it has been shown that Glatiramer Acetate is a novel, safe and effective treatment for patients with the exacerbating-remitting form of multiple sclerosis and it is the active ingredient of Copaxone™, a medicament used for the treatment of multiple sclerosis.
The process for the preparation of Glatiramer Acetate has been described in U.S. Pat. Nos. 6,048,898; 5,800,808; 5,981,589 and 3,849,550. They all employ as starting materials four N-carboxyanhydrides derived from alanine, γ-benzyl glutamate, Nε-trifluoroacetyl lysine and tyrosine. These monomers of N-carboxyanhydrides were prepared as described in the literature by the phosgene method shown in Scheme 1.

The process for the synthesis of Glatiramer Acetate is based on the polymerization of N-carboxyanhydrides of alanine 2, γ-benzyl glutamate 3, Nε-trifluoroacetyl lysine 7 and tyrosine 5, in anhydrous and cancer suspect solvent dioxane at room temperature for 24 hours using diethylamine as initiator (Scheme 2).

The deblocking of γ-benzyl groups (first deprotection) is effected by stirring the protected copolymer 8 in hydrobromic acid/acetic acid at room temperature for 17 hours. These conditions also facilitate the cleavage of the polypeptide thereby furnishing the intermediate 9.
The next step in the prior art literature process is the removal of Nε-trifluoroacetyl groups (second deprotection) of intermediate 9 by treatment with 1 M piperidine. In the final steps, Glatiramer Acetate is obtained by purification of intermediate 10 through dialysis, followed by treatment with acetic acid to form the acetate salt and by another purification by dialysis against water (Scheme 3).
Thus, the prior art literature procedure involves the polymerization of four N-carboxyanhydrides, two deprotection steps of intermediates 8 and 9 and two purification steps (Step 3 and 5 in Scheme 3) and one acetate salt formation step (Step 4 in Scheme 3).

Glatiramer acetate with the required average molecular weight (4.7 to 11 kDa) can be obtained either by chromatography of intermediate 10 containing high molecular weight species and collecting the fractions without the undesired species or by partial acid or enzymatic hydrolysis to remove the high molecular weight species with subsequent purification by dialysis or ultrafiltration. Further methods to obtain Glatiramer Acetate having the required average molecular weight are based on the preparation of the desired species while the amino acids are still protected, followed by deprotection.